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Regulation of by Exocyst-Mediated Trafficking

Noemi Polgar and Ben Fogelgren

Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813 Correspondence: [email protected]

One requirement for establishing polarity within a cell is the asymmetric trafficking of intra- cellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight- exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst are conserved in mammals, in which the specifics of exocyst-mediated traf- ficking are highly cell-type-dependent. Some exocyst members bind to certain on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establish- ment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front–back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.

symmetric membrane trafficking is a critical man 1979; Novick et al. 1980). Differential sed- Amechanism by which cell polarity is estab- imentation in a density gradient enabled the lished and maintained. It is becoming evident identification of abnormally heavy yeast cells that a large variety of eukaryotic cells can use the harboring mutations in genes critical for the octameric exocyst protein complex as a “Swiss budding of the daughter cell. Later, eight of army knife” to execute a diverse number of po- the identified genes, Sec3, Sec5, Sec6, Sec8, larized trafficking processes. Members of the Sec10, Sec15, Exo70, and Exo84 (also called exocyst complex were first identified as regula- EXOC1–8, respectively) were shown to encode tors of polarized exocytosis in the budding yeast proteins that copurified with each other, and Saccharomyces cerevisiae, during a genetic this interacting complex was named the exocyst screen of secretory mutants (Novick and Schek- (Terbush et al. 1996; Guo et al. 1999a). This 750-

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kDa holocomplex is highly conserved through- bly (Fig. 1). The exocyst belongs to the family of out the eukaryotic kingdoms and null mutants complexes associated with tethering containing of individual subunits have shown early lethal- helical rods (CATCHR), in which the subunits ity in multicellular organisms (Friedrich et al. show generally low , but 1997; Murthy et al. 2003; Fogelgren et al. 2015; have conserved helical bundles packed together Mizuno et al. 2015). into long rod-like structures (Chia and Gleeson Studies of the molecular mechanisms of 2014). Quick-freeze/deep-etch electron mi- exocyst function have been aided by emerging croscopy studies suggested that the exocyst knowledge of the exocyst’s structure and assem- subunits assemble in a side-by-side fashion,

A

Primary cilium

B ERK1/2

Tight junction Rab11 P Crumbs complex GTP Sec15

Rabin8 PAR complex Vesicle Rab8 GTP

Sec10 Adherens junction Vesicle Rab8-GDP Scribble complex

C Sec15 Sec8 Exocyst Vesicle Exo84 Sec6

Sec5 Desmosome Sec10

Par GTPases complex PIPK1γ Cytoplasm Exo70 Sec3

Site of exocytosis

Figure 1. Exocyst function in epithelial polarity. (A) The Rab11–Rabin8–Rab8 cascade facilitates Sec15 binding to the secretory vesicle. (B)PIPKg activity leads to a localized accumulation of the membrane phospholipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) (marked turquoise). By binding these phospholipids, Exo70 and Sec3 act as spatial landmarks for exocytosis at the plasma membrane. (C) The exocyst complex regulates polarity establishment and maintenance in association with GTPases, membrane phospholipids, and polarity complexes, and by trafficking secretory vesicles to several different membrane domains of epithelial cells.

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Cell Polarity and Exocyst-Mediated Trafficking

forming a T- or Y-shaped complex, in which the branes at the site of exocytosis independently of amino-terminal arms are responsible for mem- polymerization, and Exo70 could arrive to brane tethering and regulatory interactions and polarized sites via both actin-dependent and the carboxy-terminal domains pack together in -independent routes (Finger et al. 1998; Boyd parallel (Hsu et al. 1998; Matern et al. 2001; et al. 2004; Zajac et al. 2005; Liu and Novick Munson and Novick 2006). Early work in yeast 2014). Other studies of yeast and mammalian implicated Sec15 as the subunit that directly vesicle trafficking, however, suggest that the bound Rab GTPases on the surface of secretory exocyst holocomplex, including Sec3 and vesicles (Salminen and Novick 1989; Guo et al. Exo70, can be present on secretory vesicles 1999b), and Sec3 as the plasma-membrane- and that the polarized subcellular localization bound subunit and the spatial landmark for of Sec3 is dependent on an intact secretory exocyst-destined exocytosis (Finger et al. pathway and actin polymerization (Roumanie 1998). Subunit interactions of the exocyst com- et al. 2005; Bendezu and Martin 2011; Bendezu plex have been extensively studied using various et al. 2012). In addition, AP-1B, a vesicle-asso- methods in yeast and in mammals. These stud- ciated clathrin adaptor protein, which is re- ies revealed and confirmed stronger pairwise sponsible for basolateral protein sorting in ep- interactions, such as those between Sec3– ithelia, facilitated the recruitment of both Exo70 Sec5, Sec6–Sec8, and Sec10–Sec15 (Guo et al. and Sec8 to the secretory vesicle (Folsch et al. 1999a,b; Matern et al. 2001; Vegaand Hsu 2001; 2003). This finding supports the model in Munson and Novick 2006; Katoh et al. 2015; which all exocyst subunits—both Exo70- and Heider et al. 2016). Some exocyst interaction– Sec8-containing subcomplexes—are present based models proposed two four-subunit sub- on the vesicle. complex-architectures for both yeast and mam- To fulfill its tethering function following malian complexes. Here, the core module of vesicle delivery, the exocyst has to interact with Sec3, Sec5, Sec6, and Sec8 is connected to the the target membrane. This interaction is medi- vesicle-attached subcomplex of Sec10, Sec15, ated through direct binding of Sec3 and Exo70 Exo70, and Exo84 mainly through the Sec8– subunits with phosphatidylinositol(4,5)-bis- Sec10 interaction (Katoh et al. 2015; Heider phosphate (PtdIns(4,5)P2) located primarily et al. 2016). This supports previous cell frac- on the inner leaflet of the plasma membrane tionation studies showing distinct distribution (He et al. 2007; Liu et al. 2007; Zhang et al. of Sec10–Exo84 and Sec5–Sec6 cofractions in 2008; Shewan et al. 2011; Pleskot et al. 2015). rat pheochromocytoma cells (Moskalenko et al. The first studies of the exocyst in polarized ep- 2003). In mammals, several of the subunits are ithelial cells implicated the exocyst in mainly predicted to have different isoforms as a result basolateral vesicle trafficking to sites of cell– of alternative splicing (UniProt Consortium cell contacts (Grindstaff et al. 1998; Lipschutz 2015). Discussed later in this review, alternative et al. 2000). Yet, the finding that members of splicing of the exocyst genes might be a major the exocyst complex can directly bind with regulatory mechanism by which cells control PtdIns(4,5)P2, which can be located on the api- polarity and phenotype. cal surface of polarized mammalian epithelial cells (Di Paolo and De Camilli 2006; Gassama- Diagne et al. 2006; Martin-Belmonte et al. Exocyst in Trafficking and Plasma-Membrane 2007), suggested the potential that the exocyst Targeting may also take part in apical delivery under cer- Initial studies in budding yeast suggested that tain conditions. Although this exocyst–phos- six of the exocyst subunits traveled to the cell pholipid interaction may be crucial for exocytic membrane associated with exocytic vesicles events, the spatial and temporal control of exo- along actin cables, transported by the type V cyst-mediated exocytosis also hinges on regula- , Myo2p (Jin et al. 2011). Unlike the tion by known polarity complexes and various rest of the exocyst, Sec3 localized to target mem- small GTPases.

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N. Polgar and B. Fogelgren

The Exocyst’s Interaction with Polarity (Blankenship et al. 2007; Campbell et al. 2009). Determinants A recent study showed that Crumbs binds to Par6 in a Cdc42-dependent manner (Whitney Key factors in epithelial apicobasal and mesen- et al. 2016), indicating that exocyst–Par com- chymal front–back polarity are the evolu- plex interactions may be necessary for Crumbs tionarily conserved polarity protein complexes: trafficking. One study reported that the yeast Crumbs, Par, and Scribble. In epithelial tissues, Exo84 subunit binds with homologs of mam- the Crumbs and the Par complexes show an malian lethal giant larvae (Lgl), a member of the apical localization at the apical junctional com- basolateral Scribble polarity complex (Zhang plex (AJC), whereas the Scribble complex is ba- et al. 2005). However, a relationship between solateral (Pieczynski and Margolis 2011; Worz- exocyst and Scribble complex members has feld and Schwaninger 2016). In migrating cells, not been shown in non-yeast cells. The above Crumbs and Par complexes are mainly located findings indicate that the exocyst may orches- at the leading edge, but Scribble has been found trate the proper localization of each polarity at both the leading edge and the rear of migrat- complex during epithelial polarization. ing cells (Nelson 2009). One of the first reported functional interactions of the exocyst was with Exocyst Regulation by Small GTPases members of the Par complex. Early studies in yeast showed that cell division control protein Throughout polarity establishment and main- 42 (Cdc42), the small GTPase associated with tenance, cells can direct spatial and temporal the Par complex, coordinates polarized exocyst control of exocyst function through small intra- localization (Zhang et al. 2001). Biochemical cellular signal transducers from the Rab, Ral, associations were shown between exocyst sub- and Rho GTPase families. The exocyst interacts units Sec3 and Sec10 with Cdc42 in both yeast with numerous GTPases acting as their effector and mammalian cells (Zhang et al. 2001, 2008; (Lipschutz and Mostov 2002; Munson and Roumanie et al. 2005; Yamashitaet al. 2010; Zuo Novick 2006). et al. 2011). Later reports in mammalian cells The Rab family of GTPases is generally re- confirmed a biochemical interaction between sponsible for directing intracellular vesicle traf- the exocyst and other members of the Par com- ficking to specific membrane locales. On the plex: the partitioning defective-3 (Par3) and surface of yeast secretory vesicles destined for atypical protein C (aPKC) (Lalli 2009). the budding daughter cell, the interaction of However, subsequently, it was shown that the Sec15 and the Rab GTPase Sec4p is the first subunit of the Par complex that the exocyst in- regulatory step of polarized exocytosis (Guo teracted directly with was partitioning defec- et al. 1999b). In various types of animal cells, tive-6 (Par6) (Zuo et al. 2011), and this was an evidence shows that the major Rab GTPases that interaction stimulated by RalA GTPase activity use the exocyst as an effector are Rab8 (yeast (Das et al. 2014). Sec4p homolog) and Rab11. Both Rab8 and Exocyst localization also depends on the Rab11 were shown to directly bind Sec15 (Wu Crumbs polarity complex, consisting of three et al. 2005; Feng et al. 2012). Interestingly, a major proteins: Crumbs, protein associated Rab11–Rab8 signaling cascade has been de- with Lin-7 1 (PALS1), and PALS1-associated scribed. In this cascade, Rabin8, which is a tight junction protein (PATJ). PALS1 regulates Rab8 guanine exchange factor (GEF), binds tight junction (TJ) architecture and, when de- exocyst Sec15 and Rab11-GTP as well (Chiba pleted, it results in disrupted exocyst localiza- et al. 2013). Rab11, in turn, triggers Rabin8’s tion to the adherens junctions (AJs) (Wanget al. GEF activity toward Rab8 (Wu et al. 2005; 2007; Pieczynski and Margolis 2011). On the Knodler et al. 2010). Rab11’s interaction with other hand, studies of Drosophila embryonic Sec15 seems to be GTP-dependent and uses epithelium formation suggested that Exo84 is Sec15’s carboxy-terminal domain (Zhang et al. essential for the apical localization of Crumbs 2004; Wu et al. 2005). The Rab8–Rabin8–Rab11

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Cell Polarity and Exocyst-Mediated Trafficking

interactions with the exocyst define a subset of membrane expansion sites in yeast. Yet disrup- vesicles trafficking from the trans-Golgi net- tion of the Sec3–Rho1 interaction did not result work (TGN) and from the recycling endosomes in complete exocyst mislocalization, as Sec5 and to sites of exocyst-mediated exocytosis on the Exo70 appeared to be targeted correctly (Guo plasma membrane. One study in Drosophila re- et al. 2001). Additionally, although activated ported that Sec15 also bound to other Rab forms of Rho3 have been shown to bind GTPases, including Rab3 and Rab27, but not Exo70 (Robinson et al. 1999), Exo70 localiza- Rab4, Rab6, or Rab7 (Wu et al. 2005). Further tion appears to be independent of Rho3, sug- studies elucidating the role of these interactions gesting that there are parallel pathways targeting and exploring other possible regulatory Rab– the exocyst subunits during cell polarization exocyst interactions are needed. (Roumanie et al. 2005). It is possible that mem- The Ral family of GTPases regulates a di- bers of the Rho GTPase family have partially verse array of cellular processes, and contributes overlapping functions to regulate vesicle exocy- to exocyst complex assembly via direct interac- tosis acting through their shared effector, the tions with Sec5 and Exo84 (Moskalenko et al. exocyst. For example, Cdc42, another Rho 2002, 2003). Knockdown of RalA leads to either GTPases that is essential in establishing cell po- a decreased rate of exocyst assembly or a de- larity in a wide variety of cells, is also known to creased stability of the holocomplex, and dis- interact with both Sec3 and Exo70 subunits rupted RalA–Sec5 interaction results in defec- (Zhang et al. 2001; Wu et al. 2010). And whereas tive membrane protein sorting (Moskalenko in yeast, Cdc42 competes with Rho1 for binding et al. 2002). Because of an overlap in the RalA- the Sec3 amino-terminal region, the sites of binding sites for Exo84 and Sec5, these exocyst Cdc42 and Rho3 interactions were mapped to subunits bind RalA in a competitive manner different Exo70 domains (Zhang et al. 2001; Wu (Jin et al. 2005). RalA was proposed to mediate et al. 2010). Interestingly, Sec3 in higher eukary- vesicle trafficking by regulating assembly of the otes lacks the amino-terminal region necessary Sec5-containing subcomplex on the plasma for Cdc42/Rho1 binding, suggesting that Sec3 membrane, and that of the vesicle-associated may have evolved a novel Rho1/Cdc42-binding Exo84-containing subcomplex, before holo- domain, or may form the Rho- in complex assembly on the plasma membrane coordination with other exocyst components or (Moskalenko et al. 2003). On the other hand, proteins (Matern et al. 2001; Sakurai-Yageta a more recent study showed that Ral GTPases et al. 2008). were responsible for the spatial regulation of the exocyst, as reduction of RalA or RalB expression led to distinct changes in the subcellular local- THE EXOCYST IN APICOBASAL POLARITY ization of the complex, but contrary to earlier OF EPITHELIA findings, did not appear to affect exocyst assem- Exocyst and Basolateral Trafficking—The bly (Spiczka and Yeaman 2008). Forming of Cell Junctions The Rho family of GTPases also has a di- verse role in regulating cellular processes, in- Establishment and maintenance of the apico- cluding cell polarity and actin dynamics. Sec3 basal cell polarity is essential for proper epithe- and Exo70 contribute to polarized targeting of lial barrier and transport functions. Critical to the exocyst not only through their binding with establishing this polarity are specialized cell– membrane phospholipids but via interactions cell junctions, which not only function as essen- with members of the Rho GTPase family. Mu- tial components of the epithelial barrier, but tations of the Rho1 GTPase disrupted the estab- importantly act to divide the cell’s plasma lishment and maintenance of polarized exocyst membrane into structurally and functionally localization in yeast. The GTP-dependent inter- different apical and basolateral domains. There action of Rho1 with the Sec3 amino-terminal is much evidence that epithelial cells use the domain likely ensures the recruitment of Sec3 to exocyst to regulate vesicle trafficking to these

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N. Polgar and B. Fogelgren

junctions, allowing for a dynamic regulation of also may be because RalA and RalB have differ- junctional composition, assembly, and mainte- ent exocyst-binding affinities: active RalB was nance. shown to bind the exocyst less efficiently than Following initial cell–cell contact of single RalA despite their high (.80%) sequence ho- epithelial cells, the mobile plasma-membrane mology (Shipitsin and Feig 2004). Thus, RalA pool of E-cadherin rapidly translocates to activity likely plays a key role in exocyst-medi- emerging contact sites (Adams et al. 1998). E- ated epithelial polarity establishment. cadherin-mediated cell–cell adhesion triggers However, not only the proper formation of recruitment of the exocyst complex from the the AJ, but its maintenance, is dependent on cytoplasm to sites of intercellular contacts, exocyst activity. Studies in Drosophila showed and as polarity continues to develop the exocyst that a mutation of Sec5 allowed for DE-cad- complex accumulates at the AJC (Grindstaff herin endocytosis, but led to an accumulation et al. 1998; Yeaman et al. 2004). The AJC, which of DE-cadherin in the recycling endosomal includes the TJs, the AJs, and the apical polarity compartment and a failure of DE-cadherin re- complexes, connects the extracellular contacts cycling to the AJ (Langevin et al. 2005). Recent between cells with the intracellular actin cyto- evidence showed that cholera toxin interferes skeleton. with the Rab11/exocyst-mediated E-cadherin In mammalian epithelial cells, it has been trafficking to AJ, contributing to intestinal bar- well established that the exocyst binds to and rier failure in Drosophila (Guichard et al. 2013). traffics E-cadherin (Yeaman et al. 2004; Xiong In addition, toxins of Bacillus anthracis affect et al. 2012), as has also been shown for DE- the AJ formation through decreasing Rab11 cadherin, the Drosophila E-cadherin ortholog and Sec15, resulting in impaired barrier func- (Langevin et al. 2005). During AJ formation, tion both in Drosophila epithelium and human type I phosphatidylinositol-4-phosphate 5-ki- endothelial cells (Guichard et al. 2010). nase g (PIPKIg) colocalizes and binds to E-cad- Found only in vertebrates, TJs are the most herin (Akiyama et al. 2005; Ling et al. 2007), apical components of the cell–cell junctional and by generating PtdIns(4,5)P2, PIPKIg re- complexes, and are mainly composed of trans- cruits Exo70 and Sec3 to target exocyst activity membrane proteins. TJs serve as a diffusion bar- (Liu et al. 2007; Zhang et al. 2008). Although in rier that maintains cell-membrane polarity and Exo70-depleted cells E-cadherin is capable of create a semipermeable barrier that controls forming dispersed intercellular contacts, the AJ paracellular trafficking. The exocyst complex, fails to expand and form linear cohesive adhe- along with members of the Par complex, local- sions. In addition, PIPKg appears to strengthen izes to the TJs of MDCK and other polarized the association between E-cadherin and Exo70, epithelial cells (Charron et al. 2000; Lipschutz thus promoting the targeting of exocyst and its et al. 2000; Rogers et al. 2004). Ral GTPases have cargo to the forming AJ (Xiong et al. 2012). been shown to regulate TJ formation by using Another factor regulating E-cadherin traf- the exocyst as their effector, but RalA and RalB ficking through modulating exocyst function is act on the process in an opposing manner. RalA RalA GTPase. In fully polarized MDCK cells, is necessary for TJ formation through exocyto- expression of a constitutively active mutant sis and RalB regulates TJ composition via endo- RalA enhanced basolateral membrane delivery, cytosis, yet shRNA-mediated knockdown of ei- including E-cadherin, but not apical membrane ther of these GTPases had no detectable effect delivery (Shipitsin and Feig 2004). Similar ex- on overall apicobasal membrane polarity (Ha- pression of constitutively active RalB did not in- zelett et al. 2011). With respect to epithelial bar- crease the basolateral delivery of E-cadherin. rier, knockdown of the Sec10 subunit by shRNA This divergence of RalA versus RalB regulation in MDCK cells appeared to not affect the cellu- of the exocyst may bebecause of the Ral GTPases’ lar localization of ZO1, a TJ component (Zuo distinct cellular localization: RalA, but not RalB, et al. 2009; Polgar et al. 2015). It has been shown localizes to perinuclear recycling endosomes. It that Sec10 overexpression in MDCK cells led to

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Cell Polarity and Exocyst-Mediated Trafficking

increased E-cadherin synthesis and delivery, a later study, using an MDCK cell model of early and overall protection of epithelial barrier in- cystogenesis (two- and three-cell stages) in 3D tegrity (Park et al. 2010; Fogelgren et al. 2014). matrigel cultures, provided strong evidence that Therefore, follow-up analysis of TJ trafficking Rab8a used the exocyst as an effector in coordi- should be performed in more robust genetic nation with the Par complex to deliver podoca- knockout epithelial cells now that a conditional lyxin/gp135 and other apical cargo to the apical knockout mouse model is available (Fogelgren initiation domain (Bryant et al. 2010). et al. 2015). Further evidence for exocyst-mediated api- Exocyst trafficking is also important for the cal targeting has been reported in urothelial formation of another type of epithelial intercel- progenitor cells of the urinary tract (Lee et al. lular junction, the desmosomes, which form 2016), in Drosophila photoreceptor epithelial connections with the intermediate filaments of cells (Beronja et al. 2005), in Xenopus neural the . In mammalian epithelial cells, tube epithelial cells (Ossipova et al. 2014), in Sec3 was noted to colocalize with a subset of placental syncytiotrophoblast (Gonzalez et al. the exocyst complexes at the desmosome (An- 2014), and in renal collecting duct cells on dersen and Yeaman 2010). Sec6 also colocalized aquaporin vesicles (Barile et al. 2005). Addi- with desmosomal proteins in MDCK cells, tionally, in human airway epithelia in a model although pools of Sec6 were also detected in a of inflammatory airway diseases, the exocyst has nondesmosomal localization (Inamdar et al. been implicated in the apical secretion of mu- 2016). Knockdown of Sec3 did not result in an cus-component mucin 5AC (Li et al. 2015). intracellular accumulation of desmosomal des- Recent evidence shows that exocyst also lo- moglein (Dsg2) transport vesicles, but rather calizes to the primary cilium on the apical sur- Dsg2 was abnormally distributed evenly on the face of renal epithelial cells and is pivotal in cell surface, indicating successful exocytosis but primary cilia formation and trafficking (Rogers impaired desmosome targeting. Reduced Sec3 et al. 2004; Zuo et al. 2009; Fogelgren et al. 2011; expression thus disrupted the morphology of Polgar et al. 2015; Seixas et al. 2016). The pri- desmosomes, yet had no detectable effect on mary cilium is a finger-like protrusion on the AJ formation (Andersen and Yeaman 2010). cell surface that functions as a sensory and sig- naling organelle, and its formation could be considered one of the final steps in epithelial The Exocyst in the Apical Domain apicobasal polarization. Defects in primary cilia Establishment of epithelial polarity requires di- formation and function characterize numerous rected trafficking specifically to the apical plas- pathologies, termed ciliopathies (Powles-Glover ma membrane. The exocyst is most widely rec- 2014). Several of the exocyst-regulating GTPases ognized as a basolateral targeting complex, but (and their GEFs) have been shown to regulate there is growing evidence that in some epithelial primary ciliogenesis, such as Rab8, Rabin8, cell types under certain conditions, the exocyst Rab11, Cdc42, and its GEF TUBA (Knodler can also coordinate apical exocytosis. Although et al. 2010; Zuo et al. 2011; Feng et al. 2012; an early study using pulse-chase experiments in Choi et al. 2013; Baek et al. 2016), whereas dis- Sec10-overexpressing polarized MDCK cells ruption of exocyst function by silencing Sec10 showed an increase of basolateral membrane impaired both primary cilium formation and delivery of E-cadherin and not apical gp135, signaling (Zuo et al. 2009; Fogelgren et al. these cells also had a significant increase of 2011; Polgar et al. 2015). intracellular vesicles trafficking to the apical membrane and an increased apical secretion The Exocyst in the Maintenance of Apicobasal of g80 (Lipschutz et al. 2000). Also, exocyst in- Polarity hibition by function-blocking antibodies re- sulted in decreased basolateral, but not apical Although the exocyst contributes to the estab- cargo delivery (Grindstaff et al. 1998). However, lishment of apicobasal polarity through its teth-

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ering function before exocytosis, it plays a role larity proteins and the cytoskeleton, and its role in the maintenance of this polarity by mediating in polarized intracellular trafficking. endocytic and recycling events. In MDCK cells, As is the case for apicobasal polarity of epi- certain exocyst subunits localized not only to thelial cells, one of the factors that define the early endosomes, but to transferrin-positive re- front–rear polarity of migrating cells is the cycling endosomes and Rab11a-positive apical asymmetric distribution of membrane phos- recycling endosomes. Blocking exocyst function pholipids. A spatiotemporal generation and en- using Sec8 antibodies revealed that the exocyst richment of PtdIns(4,5)P2 and phosphatidyl- is necessary for basolateral and apical recycling inositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) as well as basolateral-to-apical transcytosis (Oz- characterizes the plasma membrane of the lead- tan et al. 2007). Knockdown of Rab11 or Sec15 ing edge of migrating cells (Funamoto et al. resulted in a significant decrease of exocytic 2002; Nelson 2009). As previously mentioned, events of recycling GFP-fused transferrin re- Sec3 and Exo70 interact with the phospholipid ceptor (Takahashi et al. 2012). Moreover, a fluo- PtdIns(4,5)P2, and this binding is required for rescently tagged Sec8 was found to travel to targeted exocyst-mediated exocytosis (He et al. the cell membrane on recycling vesicles, and 2007; Zhang et al. 2008). PIPKIg, a type 1 phos- Sec8 knockdown led to a recycling block phatidylinositol-phosphate kinase, regulates with a perinuclear accumulation of transferrin exocyst-mediated a5- and b1-integrin traffick- cargo (Rivera-Molina and Toomre 2013). Very ing to the leading edge of migrating cells by recently, the exocyst was identified as a network generating PtdIns(4,5)P2 and binding exocyst hub linking key components of exo- and endo- subunits (Thapa and Anderson 2012; Thapa cytic pathways (Jose et al. 2015). This is in et al. 2012). Knockdown of different exocyst support of previous data showing exocyst components led to decreased integrin delivery colocalization with different endosomal com- to the cell’s leading edge, hindering cell migra- partments (Sommer et al. 2005) and with clath- tion (Zuo et al. 2006; Spiczka and Yeaman rin and its adaptors in Drosophila (Langevin 2008; Thapa and Anderson 2012; Thapa et al. et al. 2005). 2012). This suggests that exocyst function di- rectly contributes to the mechanics of cell migration. THE EXOCYST IN FRONT–REAR POLARITY OF MIGRATORY CELLS Exocyst-Mediated Cytoskeletal Polarization Exocyst and Membrane Polarization during Front–rear polarization of migratory cells, and Cell Migration cell migration itself, involve dynamic changes in Directional cell migration is dependent on the the actin cytoskeletal structure. Budding yeast formation of front–rear polarity (Fig. 2). Sig- mutants of the late secretory pathway, including nals from the extracellular matrix and growth exocyst members Sec10 and Sec15, had disorga- factors are relayed by intracellular signaling nized actin . This was the first to trigger reorganization of the plasma mem- indication that the exocyst might affect actin brane, the cytoskeleton, and protein trafficking, organization (Aronov and Gerst 2004). Sup- which are all necessary for front–rear polarity porting these results, the mammalian Exo70 formation. Numerous signaling pathways and was shown to interact with Arpc1B, a member trafficking mechanims necessary for the estab- of the Arp2/3 complex, responsible for nucle- lishment for apicobasal polarity are also used to ating actin for the generation of the actin net- regulate front–rear polarization during cell mi- work necessary for cell migration. Exo70 acts as gration. In the following sections, we will pre- a downstream effector for Cdc42 and Rac1 sent an overview of how the exocyst contributes GTPases in this respect, stimulating the actin to front–rear polarity through plasma mem- branching necessary for filopodia and lamelli- brane reorganization, interactions with cell po- podia formation (Zuo et al. 2006; Liu et al.

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Cell Polarity and Exocyst-Mediated Trafficking

Exocyst Rab8/Rab11

Arp2/3

WAVE 2

Membrane RalA Exo70 curvature Exo70 Actin polymerization and branching

GEF-H1 Cdc42

RhoA

Sec5

Cytoplasm Integrins

Rab8/Rab11

MMPs Exocyst

Figure 2. Front–rear polarity of migratory cells. The exocyst orchestrates cytoskeletal reorganization as an effector of different small GTPases, both regulating membrane curvature and stimulating actin branching. Additionally, evidence shows the migratory cell directs the exocyst to the leading edge to participate in integrin trafficking and signaling as well as matrix metalloproteinase (MMP) secretion. Figures were prepared using the Biomedical-PPT-Toolkit-Suite (Motifolio, Sykesville, MD).

2012). Recent evidence revealed that the exocyst the cell’s leading edge (Biondini et al. 2016). subunits Exo70 and Sec6 bound with members Additionally, Exo70 contributes to Arp2/3ac- of the wave regulatory complex (WRC), another tivity and filopodia formation by generating a effector of Rac1 GTPase. When this biochemical curvature in the plasma membrane to make interaction was disrupted in HEK293 cells, their space for the forming actin network (Zhao speed of cell motility was decreased, and the et al. 2013). The Arp2/3-exocyst interaction exocyst proved to regulate WRC targeting to was shown to be regulated by external stimulat-

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N. Polgar and B. Fogelgren

ing factors, such as epidermal growth factor oral squamous cell carcinoma cells (Tanaka (EGF) in migrating HeLa cells (Zuo et al. and Iino 2015). The exocyst complex colocalizes 2006). Interestingly, Exo70 was shown to be a with microtubules and the microtubule-orga- direct substrate of the extracellular signal-regu- nizing center in the PC12 cell line, derived lated (ERK1/2), and Exo70 phosphor- from an undifferentiated pheochromocytoma ylation by ERK1/2 appears to promote exocyst (Vega and Hsu 2001). The microtubule-associ- complex assembly on EGF signaling (Ren and ated guanosine exchange factor GEF-H1 di- Guo 2012). In polarized renal epithelium, in- rectly binds with exocyst Sec5. This binding is creased exocyst activity by Sec10 overexpression promoted by RalA, and leads to subsequent ac- was linked to an increased ligand-induced EGF tivation of RhoA, which is necessary for exocyst receptor (EGFR) endocytosis and downstream complex assembly and proper localization to signaling activity that promoted exocyst assem- the leading edge (Pathak et al. 2012). bly (Fogelgren et al. 2014). It is therefore possi- ble that a similar positive feedback loop exists Polarized Intracellular Trafficking and the in migratory cells, contributing to the actin Exocyst in Migratory Cells cytoskeleton reorganization and the motile phenotype. Another aspect of front–rear orientation is the Actin organization in mammalian cells is polarization of endo- and exocytic pathways, also dependent of the Ral GTPases. RalA inter- regulating the plasma membrane proteome dis- actions with Sec5 and Exo84 have proved a key tribution in the migrating cell. Recent reports to actin cytoskeleton regulation. When these showed that the exocyst is necessary for the po- RalA–exocyst interactions are perturbed, cell larized exocytosis of matrix metalloproteinases morphological changes are observed in migra- (MMPs), a family of necessary for ex- tory cells, including defects in lamellipodia tracellular matrix degradation, aiding cell mi- formation (Hazelett and Yeaman 2012). Fur- gration and invasion. Exocyst subunits Sec3 thermore, inhibition of RalA–Sec5 binding and Sec8 were shown to directly bind IQGAP1, prevented filopodia formation triggered by in- a Rho GTPase effector, which regulates cell po- flammatory cytokine signaling (Sugihara et al. larity during migration (Noritake et al. 2005). 2002). In contrast to mesenchymal cells, in nor- This association was stimulated by GTP-bound mal rat kidney epithelial cells the exocyst was Cdc42 and RhoA GTPases. In addition, invado- shown to promote cell migration as an RalB, podial matrix degradation was affected by dis- not an RalA, effector. In this model, knockdown ruption of this interaction, as depletion of of RalB and Sec5, but not RalA, hindered cell either Sec8 or IQGAP1 led to decreased exocy- motility during wound healing. RalB GTPase tosis, but not biosynthesis of membrane-type was necessary for both exocyst localizations to MMP1 (Sakurai-Yageta et al. 2008). Further ev- the leading edge and for the assembly of the idence showed that blocking exocyst function holocomplex (Rosse et al. 2006). These results through Exo70, Exo84, or Sec8 knockdown highlight that the exocyst might contribute to abolishes, whereas Exo70 overexpression pro- the migratory phenotype downstream from dif- motes, MMP secretion. Moreover, blocking ferent regulatory signals in mesenchymal versus Exo70 phosphorylation by Erk1/2 in tumor epithelial cells. cells inhibits secretion of MMPs (Ren and Coordinated modulations of microtubules Guo 2012). The exocyst’s interaction with the and intermediate filaments are well known to endosomal Arp2/3 activator Wiskott–Aldrich regulate front–rear cell polarization and the syndrome protein and Scar homolog (WASH) migratory process itself (Kaverina and Straube also regulates focal delivery of MT1-MMP in 2011; Chung et al. 2013). At the level of inter- invasive breast carcinoma cells (Liu et al. 2009; mediate filaments, knockdown of Sec8 reduced Monteiro et al. 2013; Yamamoto et al. 2013). cytokeratin 8 phosphorylation, and subse- Showing subcellular polarization, the exo- quently suppressed cell migration in human cyst was found to accumulate at focal complexes

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Cell Polarity and Exocyst-Mediated Trafficking

forming at the leading edge, but not at focal during the process of EMT (or the reverse, mes- complexes located elsewhere on migrating cells enchymal–epithelial transition or MET), nor (Zuo et al. 2006; Spiczka and Yeaman 2008). what regulatory mechanisms distinguished ep- This association between the exocyst and pax- ithelial exocyst activity from mesenchymal exo- illin-containing focal complexes is regulated by cyst activity. the RalA and RalB GTPases, as Ral binding to In 2013, the exocyst subunit Exo70 was Sec5 is necessary for the exocyst–paxillin inter- identified as an alternative splicing target for action in cells (Spiczka and Yeaman 2008). The ESRP1, one of the keyepithelial-specific splicing exocyst also takes part in the regulation of pax- factors to regulate isoform switching of proteins illin phosphorylation. One study in migrating during EMT. Expression patterns revealed one rat kidney epithelial cells showed that the exo- Exo70 isoform to be epithelial-specific, and an- cyst and the Par complex member aPKC local- other alternative spliced isoform to be mesen- ized to the leading edge in a mutually dependent chymal-specific, with differential expression of manner (Rosse et al. 2009), and aPKC bound to these isoforms correlated with cancer progres- the exocyst via the protein Kibra. This aPKC– sion (Lu et al. 2013). Although increased ex- Kibra–exocyst complex is increased during cell pression of the epithelial-specific Exo70 isoform migration, and at the leading edge it activates can induce epithelial-like properties, the mes- JNK kinase responsible for phosphorylating enchymal-specific Exo70 isoform promotes paxillin and thus controlling the stability of fo- lamellipodia and invadopodia formation by in- cal adhesions (Rosse et al. 2009). teracting with ARP2/3 and triggering actin In addition to studies showing that the exo- branching. These results suggested that alterna- cyst contributes to integrin trafficking to the tive splicing of the exocyst members can give rise leading-edge cell membrane of migratory cells, to isoforms with distinct functions (Lu et al. there is also evidence that the exocyst helps to 2013). Cells may regulate the exocyst during regulate integrin signaling. Integrins mediate EMT not only through isoform switching but anchorage-dependent cell-cycle regulation. In by regulating the mRNA expression of mesen- the absence of integrin signal, membrane raft chymal-specific proteins, such as N-cadherin. domains that act as signaling platforms for Recent evidence indicated that the exocyst growth factor receptor signaling are internalized Sec8 regulated N-cadherin expression, (del Pozo et al. 2004). However, similar to in- but not E-cadherin, by controlling transcription tegrin-mediated adhesion, in Ras-dependent of both Smad3 and Smad4 downstream from cancers, active RalA, Arf6 GTPase, and the exo- transforming growth factor b (TGF-b) signal- cyst promote the exocystosis of raft microdo- ing (Tanaka et al. 2016). Given these recent dis- mains, contributing to anchorage-independent coveries, further studies are warranted to ex- signaling (Pawar et al. 2016). plore the differential function of exocyst isoforms, and what happens to the exocyst and associated molecules during the dynamic pro- THE EXOCYST IN EPITHELIAL– cesses of EMT and MET. MESENCHYMAL TRANSITION Epithelial–mesenchymal transition (EMT) is a CONCLUDING REMARKS global process through which polarized epithe- lial cells undergo phenotypic changes, such as Research efforts in recent years have uncovered the loss of cell–cell adhesion and apicobasal novel aspects of exocyst function and regulation polarity, and transform to acquire a motile mes- in both apicobasal and front–back cell polarity. enchymal state. EMT plays a role in numerous We have not only learned more about the exo- developmental processes, and is thought to play cyst as a tether, but gathered further evidence an important role in several pathologies as well, how the cell uses this “Swiss army knife” of a such as cancer metastasis. Until recently, noth- protein complex in a wide range of cellular pro- ing was known about how cells use the exocyst cesses necessary for polarity establishment,

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maintenance, and beyond. Yet, many more Bendezu FO, Vincenzetti V, Martin SG. 2012. Fission yeast questions remain to be answered regarding Sec3 and Exo70 are transported on actin cables and lo- calize the exocyst complex to cell poles. PLoS ONE 7: transcriptional regulation of the exocyst genes, e40248. mechanisms, and regulators of holo- and sub- Beronja S, Laprise P,Papoulas O, Pellikka M, Sisson J, Tepass complex assembly, the possible role of subunit U. 2005. Essential function of Drosophila Sec6 in apical isoforms and their effect on exocyst function exocytosis of epithelial photoreceptor cells. J Cell Biol 169: 635–646. and regulation, as well as cargo specificity. Go- Biondini M, Sadou-Dubourgnoux A, Paul-Gilloteaux P, ing forward, future studies will no doubt ad- Zago G, Arslanhan MD, Waharte F,Formstecher E, Hert- dress these and other important questions to zog M, Yu J, Guerois R, et al. 2016. Direct interaction shed light on eukaryotic exocyst function and between exocyst and Wave complexes promotes cell pro- trusions and motility. J Cell Sci 129: 3756–3769. cell polarity. Blankenship JT, Fuller MT, Zallen JA. 2007. The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelial identity. J Cell Sci 120: 3099–3110. ACKNOWLEDGMENTS Boyd C, Hughes T,Pypaert M, Novick P.2004. Vesiclescarry most exocyst subunits to exocytic sites marked by the We thank Beth Lozanoff in the Department of remaining two subunits, Sec3p and Exo70p. J Cell Biol Anatomy, Biochemistry, and Physiology at 167: 889–901. the University of Hawaii for illustrations. We Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte F, Mostov KE. 2010. A molecular net- thank Amanda Lee, Brent Fujimoto, and Jose- work for de novo generation of the apical surface and phine Napoli for their thoughtful feedback. lumen. Nat Cell Biol 12: 1035–1045. Work in the Fogelgren laboratory is funded by Campbell K, Knust E, Skaer H. 2009. Crumbs stabilises the National Institutes of Health (Grant Nos. epithelial polarity during tissue remodelling. J Cell Sci 122: 2604–2612. K01DK087852, R03DK100738, P20GM103456- Charron AJ, Nakamura S, Bacallao R, Wandinger-Ness A. 06A1-8293 to B.F.) and March of Dimes (Basil 2000. Compromised cytoarchitecture and polarized traf- O’Connor Starter Scholar Research Award, ficking in autosomal dominant polycystic kidney disease. Grant No. 5-FY14-56 to B.F.). J Cell Biol 149: 111–124. Chia PZ, Gleeson PA. 2014. Membrane tethering. F1000Prime Rep 6: 74. Chiba S, Amagai Y, Homma Y, Fukuda M, Mizuno K. 2013. REFERENCES NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by switching binding specificity from phos- Adams CL, Chen YT, Smith SJ, Nelson WJ. 1998. Mecha- phatidylserine to Sec15. EMBO J 32: 874–885. nisms of epithelial cell–cell adhesion and cell compac- Choi SY,Chacon-Heszele MF,Huang L, McKenna S, Wilson tion revealed by high-resolution tracking of E-cadherin- green fluorescent protein. J Cell Biol 142: 1105–1119. FP, Zuo X, Lipschutz JH. 2013. Cdc42 deficiency causes ciliary abnormalities and cystic kidneys. J Am Soc Nephrol Akiyama C, Shinozaki-Narikawa N, Kitazawa T, Hamakubo 24: 1435–1450. T, Kodama T, Shibasaki Y. 2005. Phosphatidylinositol-4- phosphate 5-kinase g is associated with cell–cell junction Chung BM, Rotty JD, Coulombe PA. 2013. Networking ga- in A431 epithelial cells. Cell Biol Int 29: 514–520. lore: Intermediate filaments and cell migration. Curr Opin Cell Biol 25: 600–612. Andersen NJ, Yeaman C. 2010. Sec3-containing exocyst complex is required for desmosome assembly in mam- Das A, Gajendra S, Falenta K, Oudin MJ, Peschard P,Feng S, malian epithelial cells. Mol Biol Cell 21: 152–164. Wu B, Marshall CJ, Doherty P, Guo W, et al. 2014. RalA Aronov S, Gerst JE. 2004. Involvement of the late secretory promotes a direct exocyst–Par6 interaction to regulate pathway in actin regulation and mRNA transport in yeast. polarity in neuronal development. J Cell Sci 127: 686– J Biol Chem 279: 36962–36971. 699. Baek JI, Kwon SH, Zuo X, Choi SY, Kim SH, Lipschutz JH. del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, An- 2016. binding protein (Tuba) deficiency inhib- derson RG, Schwartz MA. 2004. Integrins regulate Rac its ciliogenesis and nephrogenesis in vitro and in vivo. targeting by internalization of membrane domains. Sci- J Biol Chem 291: 8632–8643. ence 303: 839–842. Barile M, Pisitkun T,Yu MJ, Chou CL, Verbalis MJ, Shen RF, Di Paolo G, De Camilli P. 2006. Phosphoinositides in cell Knepper MA. 2005. Large scale protein identification in regulation and membrane dynamics. Nature 443: 651– intracellular aquaporin-2 vesicles from renal inner med- 657. ullary collecting duct. Mol Cell Proteom 4: 1095–1106. Feng S, Knodler A, Ren J, Zhang J, Zhang X, Hong Y,Huang Bendezu FO, Martin SG. 2011. Actin cables and the exocyst S, Peranen J, Guo W. 2012. A Rab8 guanine nucleotide form two independent morphogenesis pathways in the exchange factor–effector interaction network regulates fission yeast. Mol Biol Cell 22: 44–53. primary ciliogenesis. J Biol Chem 287: 15602–15609.

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Cell Polarity and Exocyst-Mediated Trafficking

Finger FP, Hughes TE, Novick P. 1998. Sec3p is a spatial Hazelett CC, Yeaman C. 2012. Sec5 and Exo84 mediate dis- landmark for polarized secretion in budding yeast. Cell tinct aspects of RalA-dependent cell polarization. PLoS 92: 559–571. ONE 7: e39602. Fogelgren B, Lin SY,Zuo X, Jaffe KM, Park KM, Reichert RJ, Hazelett CC, Sheff D, YeamanC. 2011. RalA and RalB differ- Bell PD, Burdine RD, Lipschutz JH. 2011. The exocyst entially regulate development of epithelial tight junc- protein Sec10 interacts with Polycystin-2 and knockdown tions. Mol Biol Cell 22: 4787–4800. causes PKD-phenotypes. PLoS Genet 7: e1001361. He B, Xi F, Zhang X, Zhang J, Guo W. 2007. Exo70 interacts Fogelgren B, Zuo X, Buonato JM, Vasilyev A, Baek JI, Choi with phospholipids and mediates the targeting of the SY, Chacon-Heszele MF, Palmyre A, Polgar N, Drum- exocyst to the plasma membrane. EMBO J 26: 4053– mond I, et al. 2014. Exocyst Sec10 protects renal tubule 4065. cells from injury by EGFR/MAPK activation and effects Heider MR, Gu M, Duffy CM, Mirza AM, Marcotte LL, on endocytosis. Am J Physiol Renal Physiol 307: F1334– Walls AC, Farrall N, Hakhverdyan Z, Field MC, Rout F1341. MP, et al. 2016. Subunit connectivity, assembly determi- Fogelgren B, Polgar N, Lui VH, Lee AJ, Tamashiro KK, Na- nants and architecture of the yeast exocyst complex. Nat poli JA, Walton CB, Zuo X, Lipschutz JH. 2015. Urothe- Struct Mol Biol 23: 59–66. lial defects from targeted inactivation of exocyst Sec10 in Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller mice cause ureteropelvic junction obstructions. PLoS RH. 1998. Subunit composition, protein interactions, ONE 10: e0129346. and structures of the mammalian brain sec6/8 complex Folsch H, Pypaert M, Maday S, Pelletier L, Mellman I. 2003. and septin filaments. 20: 1111–1122. The AP-1A and AP-1B clathrin adaptor complexes define Inamdar SM, Hsu SC, Yeaman C. 2016. Probing functional biochemically and functionally distinct membrane do- changes in exocyst configuration with monoclonal anti- mains. J Cell Biol 163: 351–362. bodies. Front Cell Dev Biol 4: 51. Friedrich GA, Hildebrand JD, Soriano P.1997. The secretory Jin R, Junutula JR, Matern HT, Ervin KE, Scheller RH, protein Sec8 is required for paraxial mesoderm forma- Brunger AT. 2005. Exo84 and Sec5 are competitive regu- tion in the mouse. Dev Biol 192: 364–374. latory Sec6/8 effectors to the RalA GTPase. EMBO J 24: Funamoto S, Meili R, Lee S, Parry L, Firtel RA. 2002. Spatial 2064–2074. and temporal regulation of 3-phosphoinositides by PI 3- Jin Y, Sultana A, Gandhi P, Franklin E, Hamamoto S, Khan kinase and PTEN mediates chemotaxis. Cell 109: 611– 623. AR, Munson M, Schekman R, WeismanLS. 2011. Myosin V transports secretory vesicles via a Rab GTPase cascade Gassama-Diagne A, Yu W, ter Beest M, Martin-Belmonte F, and interaction with the exocyst complex. Dev Cell 21: Kierbel A, Engel J, Mostov K. 2006. Phosphatidylinositol- 1156–1170. 3,4,5-trisphosphate regulates the formation of the baso- lateral plasma membrane in epithelial cells. Nat Cell Biol Jose M, Tollis S, Nair D, Mitteau R, Velours C, Massoni- 8: 963–970. Laporte A, Royou A, Sibarita JB, McCusker D. 2015. A quantitative imaging-based screen reveals the exocyst as a Gonzalez IM, Ackerman WEt, Vandre DD, Robinson JM. network hub connecting endocytosis and exocytosis. Mol 2014. Exocyst complex protein expression in the human Biol Cell 26: 2519–2534. placenta. Placenta 35: 442–449. Katoh Y, Nozaki S, Hartanto D, Miyano R, Nakayama K. Grindstaff KK, YeamanC, Anandasabapathy N, Hsu S, Rod- 2015. Architectures of multisubunit complexes revealed riguez-Boulan R, Scheller RH, Nelson WJ. 1998. Sec6/8 by a visible immunoprecipitation assay using fluorescent complex is recruited to cell–cell contacts and specifies fusion proteins. J Cell Sci 128: 2351–2362. transport vesicle delivery to the basal–lateral membrane in epithelial cells. Cell 93: 731–740. Kaverina I, Straube A. 2011. Regulation of cell migration by dynamic microtubules. Semin Cell Dev Biol 22: 968–974. Guichard A, McGillivray SM, Cruz-Moreno B, van Sorge NM, Nizet V, Bier E. 2010. Anthrax toxins cooperatively Knodler A, Feng S, Zhang J, Zhang X, Das A, Peranen J, Guo inhibit endocytic recycling by the Rab11/Sec15 exocyst. W. 2010. Coordination of Rab8 and Rab11 in primary Nature 467: 854–858. ciliogenesis. Proc Natl Acad Sci 107: 6346–6351. Guichard A, Cruz-Moreno B, Aguilar B, van Sorge NM, Lalli G. 2009. RalA and the exocyst complex influence neu- Kuang J, Kurkciyan AA, Wang Z, Hang S, Pineton de ronal polarity through PAR-3 and aPKC. J Cell Sci 122: Chambrun GP, McCole DF, et al. 2013. Cholera toxin 1499–1506. disrupts barrier function by inhibiting exocyst-mediated Langevin J, Morgan MJ, Sibarita JB, Aresta S, Murthy M, trafficking of host proteins to intestinal cell junctions. Schwarz T, Camonis J, Bellaiche Y. 2005. Drosophila exo- Cell Host Microbe 14: 294–305. cyst components Sec5, Sec6, and Sec15 regulate DE-cad- Guo W, Grant A, Novick P. 1999a. Exo84p is an exocyst herin trafficking from recycling endosomes to the plasma protein essential for secretion. J Biol Chem 274: 23558– membrane. Dev Cell 9: 365–376. 23564. Lee AJ, Polgar N, Napoli JA, Lui VH, Tamashiro KK, Fuji- Guo W, Roth D, Walch-Solimena C, Novick P. 1999b. The moto BA, Thompson KS, Fogelgren B. 2016. Fibroproli- exocyst is an effector for Sec4p, targeting secretory vesi- ferative response to urothelial failure obliterates the ure- cles to sites of exocytosis. EMBO J 18: 1071–1080. ter lumen in a mouse model of prenatal congenital Guo W, Tamanoi F, Novick P.2001. Spatial regulation of the obstructive nephropathy. Sci Rep 6: 31137. exocyst complex by Rho1 GTPase. Nat Cell Biol 3: 353– Li Q, Li N, Liu CY, Xu R, Kolosov VP, Perelman JM, Zhou 360. XD. 2015. Ezrin/exocyst complex regulates mucin 5AC

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a031401 13 Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press

N. Polgar and B. Fogelgren

secretion induced by neutrophil elastase in human airway Murthy M, Garza D, Scheller RH, Schwarz TL. 2003. Muta- epithelial cells. Cell Physiol Biochem 35: 326–338. tions in the exocyst component Sec5 disrupt neuronal Ling K, Bairstow SF, Carbonara C, Turbin DA, Huntsman membrane traffic, but neurotransmitter release persists. DG, Anderson RA. 2007. Type Ig phosphatidylinositol Neuron 37: 433–447. phosphate kinase modulates adherens junction and E- Nelson WJ. 2009. Remodeling epithelial cell organization: cadherin trafficking via a direct interaction with m1B Transitions between front–rear and apical-basal polarity. adaptin. J Cell Biol 176: 343–353. Cold Spring Harb Perspect Biol 1: a000513. Lipschutz JH, Mostov KE. 2002. Exocytosis: The many mas- Noritake J, Watanabe T, Sato K, Wang S, Kaibuchi K. 2005. ters of the exocyst. Curr Biol 12: R212–R214. IQGAP1: A key regulator of adhesion and migration. Lipschutz JH, Guo W, O’Brien LE, Nguyen YH, Novick P, J Cell Sci 118: 2085–2092. Mostov KE. 2000. Exocyst is involved in cystogenesis and Novick P, Schekman R. 1979. Secretion and cell-surface tubulogenesis and acts by modulating synthesis and de- growth are blocked in a temperature-sensitive mutant livery of basolateral plasma membrane and secretory pro- of Saccharomyces cerevisiae. Proc Natl Acad Sci 76: teins. Mol Biol Cell 11: 4259–4275. 1858–1862. Liu D, Novick P.2014. Bem1p contributes to secretory path- Novick P, Field C, Schekman R. 1980. Identification of 23 way polarization through a direct interaction with complementation groups required for post-translational Exo70p. J Cell Biol 207: 59–72. events in the yeast secretory pathway. Cell 21: 205–221. Liu J, Zuo X, Yue P,Guo W. 2007. Phosphatidylinositol 4,5- Ossipova O, Kim K, Lake BB, Itoh K, Ioannou A, Sokol SY. bisphosphate mediates the targeting of the exocyst to the 2014. Role of Rab11 in planar cell polarity and apical plasma membrane for exocytosis in mammalian cells. constriction during vertebrate neural tube closure. Nat Mol Biol Cell 18: 4483–4492. Commun 5: 3734. Liu J, Yue P,Artym VV,Mueller SC, Guo W.2009. The role of Oztan A, Silvis M, Weisz OA, Bradbury NA, Hsu SC, Gold- the exocyst in matrix metalloproteinase secretion and enring JR, Yeaman C, Apodaca G. 2007. Exocyst require- actin dynamics during tumor cell invadopodia forma- ment for endocytic traffic directed toward the apical and tion. Mol Biol Cell 20: 3763–3771. basolateral poles of polarized MDCK cells. Mol Biol Cell Liu J, Zhao Y,Sun Y,He B, Yang C, Svitkina T, Goldman YE, 18: 3978–3992. Guo W. 2012. Exo70 stimulates the Arp2/3 complex for Park KM, Fogelgren B, Zuo X, Kim J, Chung DC, Lipschutz lamellipodia formation and directional cell migration. JH. 2010. Exocyst Sec10 protects epithelial barrier integ- Curr Biol 22: 1510–1515. rity and enhances recovery following oxidative stress, by Lu H, Liu J, Liu S, Zeng J, Ding D, Carstens RP,Cong Y,Xu X, activation of the MAPK pathway. Am J Physiol Renal Guo W. 2013. Exo70 isoform switching upon epithelial– Physiol 298: F818–F826. mesenchymal transition mediates cancer cell invasion. Pathak R, Delorme-Walker VD, Howell MC, Anselmo AN, Dev Cell 27: 560–573. White MA, Bokoch GM, Dermardirossian C. 2012. The Martin-Belmonte F, Gassama A, Datta A, Yu W, Rescher U, microtubule-associated Rho activating factor GEF-H1 Gerke V, Mostov K. 2007. PTEN-mediated apical segre- interacts with exocyst complex to regulate vesicle traffic. gation of phosphoinositides controls epithelial morpho- Dev Cell 23: 397–411. genesis through Cdc42. Cell 128: 383–397. Pawar A, Meier JA, Dasgupta A, Diwanji N, Deshpande N, Matern HT, Yeaman C, Nelson WJ, Scheller RH. 2001. The Saxena K, Buwa N, Inchanalkar S, Schwartz MA, Balasu- / Sec6 8 complex in mammalian cells: Characterization of bramanian N. 2016. Ral-Arf6 crosstalk regulates Ral de- mammalian Sec3, subunit interactions, and expression of pendent exocyst trafficking and anchorage independent subunits in polarized cells. Proc Natl Acad Sci 98: 9648– growth signalling. Cell Signal 28: 1225–1236. 9653. Pieczynski J, Margolis B. 2011. Protein complexes that con- Mizuno S, Takami K, Daitoku Y, Tanimoto Y, Dinh TT, trol renal epithelial polarity. Am J Physiol Renal Physiol Mizuno-Iijima S, Hasegawa Y, Takahashi S, Sugiyama F, 300: F589–F601. Yagami K. 2015. Peri-implantation lethality in mice car- rying megabase-scale deletion on 5qc3.3 is caused by Pleskot R, Cwiklik L, Jungwirth P, Zarsky V, Potocky M. Exoc1 null mutation. Sci Rep 5: 13632. 2015. Membrane targeting of the yeast exocyst complex. Biochim Biophys Acta 1848: 1481–1489. Monteiro P,Rosse C, Castro-Castro A, Irondelle M, Lagoutte E, Paul-Gilloteaux P, Desnos C, Formstecher E, Darchen Polgar N, Lee AJ, Lui VH, Napoli JA, Fogelgren B. 2015. The F, Perrais D, et al. 2013. Endosomal WASH and exocyst exocyst gene Sec10 regulates renal epithelial monolayer complexes control exocytosis of MT1-MMP at invado- homeostasis and apoptotic sensitivity. Am J Physiol Cell podia. J Cell Biol 203: 1063–1079. Physiol 309: C190–C201. Moskalenko S, Henry DO, Rosse C, Mirey G, Camonis JH, Powles-Glover N. 2014. Cilia and ciliopathies: Classic exam- White MA. 2002. The exocyst is a Ral effector complex. ples linking phenotype and genotype—An overview. Re- Nat Cell Biol 4: 66–72. prod Toxicol 48: 98–105. Moskalenko S, Tong C, Rosse C, Mirey G, Formstecher E, Ren J, Guo W. 2012. ERK1/2 regulate exocytosis through Daviet L, Camonis J, White MA. 2003. Ral GTPases reg- direct phosphorylation of the exocyst component Exo70. ulate exocyst assembly through dual subunit interactions. Dev Cell 22: 967–978. J Biol Chem 278: 51743–51748. Rivera-Molina F, Toomre D. 2013. Live-cell imaging of exo- Munson M, Novick P.2006. The exocyst defrocked, a frame- cyst links its spatiotemporal dynamics to various stages work of rods revealed. Nat Struct Mol Biol 13: 577–581. of vesicle fusion. J Cell Biol 201: 673–680.

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Cell Polarity and Exocyst-Mediated Trafficking

Robinson NG, Guo L, Imai J, Toh EA, Matsui Y, Tamanoi F. Tanaka T, Goto K, Iino M. 2016. Sec8 modulates TGF-b 1999. Rho3 of Saccharomyces cerevisiae, which regulates induced EMT by controlling N-cadherin via regulation the actin cytoskeleton and exocytosis, is a GTPase which of Smad3/4. Cell Signal 29: 115–126. interacts with Myo2 and Exo70. Mol Cell Biol 19: 3580– TerbushDR, Maurice T,Roth D, Novick P.1996. The exocyst 3587. is a multiprotein complex required for exocytosis in Sac- Rogers KK, Wilson PD, Snyder RW, Zhang X, Guo W, Bur- charomyces cerevisiae. EMBO J 15: 6483–6494. row CR, Lipschutz JH. 2004. The exocyst localizes to the Thapa N, Anderson RA. 2012. PIP2 signaling, an integrator primary cilium in MDCK cells. Biochem Biophys Res of cell polarity and vesicle trafficking in directionally mi- Commun 319: 138–143. grating cells. Cell Adh Migr 6: 409–412. Rosse C, Hatzoglou A, Parrini MC, White MA, Chavrier P, Thapa N, Sun Y,Schramp M, Choi S, Ling K, Anderson RA. Camonis J. 2006. RalB mobilizes the exocyst to drive cell 2012. Phosphoinositide signaling regulates the exocyst migration. Mol Cell Biol 26: 727–734. complex and polarized integrin trafficking in direction- ally migrating cells. Dev Cell 22: 116–130. Rosse C, Formstecher E, Boeckeler K, Zhao Y, Kremer- skothen J, White MD, Camonis JH, Parker PJ. 2009. An UniProt Consortium. 2015. UniProt: A hub for protein in- aPKC-exocyst complex controls paxillin phosphorylation formation. Nucleic Acids Res 43: D204–D212. and migration through localised JNK1 activation. PLoS Vega IE, Hsu SC. 2001. The exocyst complex associates with Biol 7: e1000235. microtubules to mediate vesicle targeting and neurite outgrowth. J Neurosci 21: 3839–3848. Roumanie O, Wu H, Molk JN, Rossi G, Bloom K, Brennwald P. 2005. Rho GTPase regulation of exocytosis in yeast is Wang Q, Chen XW, Margolis B. 2007. PALS1 regulates E- independent of GTP hydrolysis and polarization of the cadherin trafficking in mammalian epithelial cells. Mol exocyst complex. J Cell Biol 170: 583–594. Biol Cell 18: 874–885. Whitney DS, Peterson FC, Kittell AW, Egner JM, Prehoda Sakurai-YagetaM, Recchi C, Le Dez G, Sibarita JB, Daviet L, KE, Volkman BF. 2016. Binding of Crumbs to the Par-6 Camonis J, D’Souza-Schorey C, Chavrier P. 2008. The CRIB-PDZ module is regulated by Cdc42. Biochemistry interaction of IQGAP1 with the exocyst complex is re- 10: 1455–1461. quired for tumor cell invasion downstream of Cdc42 and RhoA. J Cell Biol 181: 985–998. Worzfeld T, Schwaninger M. 2016. Apicobasal polarity of brain endothelial cells. J Cereb Blood Flow Metab 36: Salminen A, Novick PJ. 1989. The Sec15 protein responds to 340–362. the function of the GTP binding protein, Sec4, to control Wu S, Mehta SQ, Pichaud F, Bellen HJ, Quiocho FA. 2005. vesicular traffic in yeast. J Cell Biol 109: 1023–1036. Sec15 interacts with Rab11 via a novel domain and affects Seixas C, Choi SY,Polgar N, Umberger NL, East MP,Zuo X, Rab11 localization in vivo. Nat Struct Mol Biol 12: 879– Moreiras H, Ghossoub R, Benmerah A, Kahn RA, et al. 885. 2016. Arl13b and the exocyst interact synergistically in Wu H, Turner C, Gardner J, Temple B, Brennwald P. 2010. ciliogenesis. Mol Biol Cell 27: 308–320. The Exo70 subunit of the exocyst is an effector for both Shewan A, Eastburn DJ, Mostov K. 2011. Phosphoinositides Cdc42 and Rho3 function in polarized exocytosis. Mol in cell architecture. Cold Spring Harbor Perspect Biol 3: Biol Cell 21: 430–442. a004796. Xiong X, Xu Q, Huang Y,Singh RD, Anderson R, Leof E, Hu Shipitsin M, Feig LA. 2004. RalA but not RalB enhances J, Ling K. 2012. An association between type Ig PI4P 5- polarized delivery of membrane proteins to the basolat- kinase and Exo70 directs E-cadherin clustering and epi- eral surface of epithelial cells. Mol Cell Biol 24: 5746– thelial polarization. Mol Biol Cell 23: 87–98. 5756. Yamamoto A, Kasamatsu A, Ishige S, Koike K, Saito K, Sommer B, Oprins A, Rabouille C, Munro S. 2005. The Kouzu Y, Koike H, Sakamoto Y, Ogawara K, Shiiba M, exocyst component Sec5 is present on endocytic vesicles et al. 2013. Exocyst complex component Sec8: A pre- in the oocyte of Drosophila melanogaster. J Cell Biol 169: sumed component in the progression of human oral 953–963. squamous-cell carcinoma by secretion of matrix met- alloproteinases. J Cancer Res Clin Oncol 139: 533– Spiczka KS, Yeaman C. 2008. Ral-regulated interaction be- 542. tween Sec5 and paxillin targets Exocyst to focal complex- Yamashita M, Kurokawa K, Sato Y, Yamagata A, Mimura H, es during cell migration. J Cell Sci 121: 2880–2891. Yoshikawa A, Sato K, Nakano A, Fukai S. 2010. Structural Sugihara K, Asano S, Tanaka K, Iwamatsu A, Okawa K, Ohta basis for the Rho- and phosphoinositide-dependent lo- Y. 2002. The exocyst complex binds the small GTPase calization of the exocyst subunit Sec3. Nat Struct Mol Biol RalA to mediate filopodia formation. Nat Cell Biol 4: 17: 180–186. 73–78. Yeaman C, Grindstaff KK, Nelson WJ. 2004. Mechanism of Takahashi S, Kubo K, Waguri S, Yabashi A, Shin HW, Katoh recruiting Sec6/8 (exocyst) complex to the apical junc- Y, Nakayama K. 2012. Rab11 regulates exocytosis of re- tional complex during polarization of epithelial cells. cycling vesicles at the plasma membrane. J Cell Sci 125: J Cell Sci 117: 559–570. 4049–4057. Zajac A, Sun X, Zhang J, Guo W.2005. Cyclical regulation of Tanaka T, Iino M. 2015. Sec8 regulates cytokeratin8 phos- the exocyst and cell polarity determinants for polarized phorylation and cell migration by controlling the ERK cell growth. Mol Biol Cell 16: 1500–1512. and p38 MAPK signalling pathways. Cell Signal 27: Zhang X, Bi E, Novick P,Du L, Kozminski KG, Lipschutz JH, 1110–1119. Guo W. 2001. Cdc42 interacts with the exocyst and reg-

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N. Polgar and B. Fogelgren

ulates polarized secretion. J Biol Chem 276: 46745– Zhao Y, Liu J, Yang C, Capraro BR, Baumgart T, Bradley RP, 46750. Ramakrishnan N, Xu X, Radhakrishnan R, Svitkina T, et Zhang XM, Ellis S, Sriratana A, Mitchell CA, Rowe T. 2004. al. 2013. Exo70 generates membrane curvature for mor- Sec15 is an effector for the Rab11 GTPase in mammalian phogenesis and cell migration. Dev Cell 26: 266–278. cells. J Biol Chem 279: 43027–43034. Zuo X, Zhang J, Zhang Y, Hsu SC, Zhou D, Guo W. 2006. Zhang X, Wang P,Gangar A, Zhang J, Brennwald P,TerBush Exo70 interacts with the Arp2/3 complex and regulates D, Guo W.2005. Lethal giant larvae proteins interact with cell migration. Nat Cell Biol 8: 1383–1388. the exocyst complex and are involved in polarized exocy- Zuo X, Guo W, Lipschutz JH. 2009. The exocyst protein tosis. J Cell Biol 170: 273–283. Sec10 is necessary for primary ciliogenesis and cystogen- Zhang X, Orlando K, He B, Xi F, Zhang J, Zajac A, Guo W. esis in vitro. Mol Biol Cell 20: 2522–2529. 2008. Membrane association and functional regulation Zuo X, Fogelgren B, Lipschutz JH. 2011. The small GTPase of Sec3 by phospholipids and Cdc42. J Cell Biol 180: Cdc42 is necessary for primary ciliogenesis in renal tu- 145–158. bular epithelial cells. J Biol Chem 286: 22469–22477.

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Regulation of Cell Polarity by Exocyst-Mediated Trafficking

Noemi Polgar and Ben Fogelgren

Cold Spring Harb Perspect Biol published online March 6, 2017

Subject Collection Cell Polarity

Regulation of Cell Polarity by Exocyst-Mediated The Crumbs3 Polarity Protein Trafficking Ben Margolis Noemi Polgar and Ben Fogelgren Phosphoinositides and Membrane Targeting in Microtubule Motors in Establishment of Epithelial Cell Polarity Cell Polarity Gerald R. Hammond and Yang Hong Geri Kreitzer and Monn Monn Myat Trafficking Ion Transporters to the Apical Role of Polarity Proteins in the Generation and Membrane of Polarized Intestinal Enterocytes Organization of Apical Surface Protrusions Amy Christine Engevik and James R. Goldenring Gerard Apodaca Signaling Networks in Epithelial Tube Formation Polarized Exocytosis Ilenia Bernascone, Mariam Hachimi and Fernando Jingwen Zeng, Shanshan Feng, Bin Wu, et al. Martin-Belmonte Making Heads or Tails of It: Cell−Cell Adhesion in Regulation of Transporters and Channels by Cellular and Supracellular Polarity in Collective Membrane-Trafficking Complexes in Epithelial Migration Cells Jan-Hendrik Venhuizen and Mirjam M. Zegers Curtis T. Okamoto Laminins in Epithelial Cell Polarization: Old Membrane Transport across Polarized Epithelia Questions in Search of New Answers Maria Daniela Garcia-Castillo, Daniel J.-F. Karl S. Matlin, Satu-Marja Myllymäki and Aki Chinnapen and Wayne I. Lencer Manninen Epithelial Morphogenesis during Liver Mechanisms of Cell Polarity−Controlled Epithelial Development Homeostasis and Immunity in the Intestine Naoki Tanimizu and Toshihiro Mitaka Leon J. Klunder, Klaas Nico Faber, Gerard Dijkstra, et al. Targeting the Mucosal Barrier: How Pathogens The Biology of Ciliary Dynamics Modulate the Cellular Polarity Network Kuo-Shun Hsu, Jen-Zen Chuang and Ching-Hwa Travis R. Ruch and Joanne N. Engel Sung

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